1. Field of the Invention
This invention relates to a procedure for high pH washing and depyrogenation of silica-based anion-exchange chromatographic sorbents.
2. Description of the Previously Published Art
In preparative or process scale production of injectables such as peptides, proteins, and pharmaceuticals the product must be purified to meet various quality control and regulatory specifications. Column chromatography is often used in large part to purify these products.
One critical issue regulating injectables is the level of pyrogen contamination in the product. Pyrogens, or fever inducing substances, are widely diverse materials ranging from inorganic to organic compounds. The pyrogens of specific concern in genetically engineered or biologically produced injectables are anionic lipo-polysaccharide bacterial cell wall components. The level of contamination by these bacterial cell wall pyrogens is strictly regulated by the Food and Drug Administration.
To maintain pyrogen levels in chromatographically purified products below FDA approved limits, organic resin based anion-exchange sorbents are routinely depyrogenated or washed with high pH caustic solutions. These washes are normally specified as ranging between 0.1 and 0.5N NaOH (pH 13-13.7). These washes are accepted as effective cleaning methods for depyrogenation of chromatographic sorbents.
High performance silica-based chromatographic sorbents can not be washed with this standard procedure due to the silica support's extremely high solubility at these alkaline pH's. The chromatographic bed rapidly deteriorates, causing channelling, loss of resolution and loading capacity and ultimately fouling of the bed which prevents passage of solutions through the column even at very high pressure. Since silica based anion-exchange sorbents cannot be cleaned and depyrogenated using standard high pH washes, the use of these media for preparation of injectables has been extremely limited.
High performance silica-based chromatographic sorbents can differ significantly from organic resin based chromatographic sorbents in their composition which can lead to confusion in terminology. The chromatographically active particles hereafter are referred to as the sorbent or packing material. In the case of high performance chromatographic sorbents, the particles usually comprise an inorganic support, often porous, which is modified with an appropriate organic coating or stationary phase to confer the chromatographic properties to the sorbent.
Large-scale column chromatography using "soft-gel" or organic resin based chromatographic sorbents has been widely used for purification of injectables despite several limitations imposed by the poor mechanical strength of these column packing materials. Column beds are susceptible to compression and plugging under high flow rates, high particle attrition during stirring, severe bed shrinkage and swelling during solvent changes or gradient elution, low loading capacity for high molecular weight compounds and relatively low resolution separations due to the large particle diameter packings. Inorganic, or silica-based chromatographic sorbents by virtue of their superior mechanical strength and wide pore structure overcome virtually all of these soft-gel limitations.
One advantage provided by organic resin based sorbents is chemical stability to a variety of solutions, including aqueous solutions in the pH range from 2-14. This allows the use of very high pH solutions (pH 13-14) to clean and depyrogenate the chromatographic packing material. Siliceous supports are known to dissolve and deteriorate rapidly when washed with solutions above pH 8.
Three general approaches have been used to attempt to extend the useful upper pH limit of silica-based chromatographic sorbents.
1. Polymeric organic coatings have been used to simultaneously confer the required chromatographic functional groups and protect the silica surface during exposure to high pH. U.S. Pat. No. 4,245,005 and Alpert and Regnier, J. Chromatogr., 185, 375-392 (1979) teach that adsorbed, crosslinked coatings of polyethyleneimine on silica, controlled porosity glass and related inorganic supports exhibit improved stability in aqueous solutions relative to the uncoated support. The upper pH limit on these types of packing materials is about pH 9.
2. Treatment of the silica particle with metal oxides, hydroxides, etc. to confer improved pH stability to the inorganic support. It is well known that the adsorption on silica of alumina and other metal oxides and hydroxides can confer improved pH stability to the silica particle. Iler in J. Colloid and Inter. Sci, 43, 399-408, 1973 shows that in static systems at pH 8 colloidal silica is stabilized in the presence of aluminum ions. The effectiveness of the stabilization is related to the proportions of silica and alumina on the surface at equilibrium. U.S. Pat. No. 4,600,646 relates to the production of a surface stabilized porous silica having a discontinuous metal oxide layer over the silica. The protected support particles and chromatographic packings thus produced have enhanced pH stability in chromatographic eluents to a maximum practical value of pH 9. U.S. Pat. No. 4,648,975 relates to a process of using silica-based sorbents modified with various metal oxides, hydroxides and related materials to carry out chromatographic separations at pH's above the normal upper limit of pH 8. The preferred pH range stated in the claims is pH 8-10. This invention does not describe an extremely high pH washing protocol acceptable for cleaning and depyrogenation of chromatographic columns.
3. Guard columns of silica particles have been used to pre-saturate the solvent stream with dissolved silica to suppress dissolution of the silica in the chromatographic column. This technique is described by Atwood et al in J. Chromatogr., 171, 109-115 (1979) and is well known to practitioners of the chromatographic art. The upper pH limit when this technique is employed is about pH 9.
As discussed above, previous attempts to stabilize silica-based chromatographic sorbents to extend the useful upper pH range above pH 8 do provide some protection of the support for chromatographic separations carried out in the pH range from 8-10. All of the above approaches fail to provide adequate protection when extremely high (pH 13-14) washes are used to clean and depyrogenate the chromatographic column due to the extremely high silica solubility (technique 1 and 3) or high leaching rates of applied metal oxide type coatings (technique 2) at these extreme pH's.